Bottom Line:
However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT.We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia.These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR.

ABSTRACTRadiation therapy (RT) is a widely accepted treatment strategy for many central nervous system (CNS) pathologies. However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT. The pathophysiology and molecular alterations that culminate in the long-term detrimental effects of CR are poorly understood, though it is thought that endothelial injury plays a pivotal role in triggering cranial injury. We therefore explored the contribution of bone marrow derived cells (BMDCs) in their capacity to repair and contribute to neo-vascularization following CR. Using high-resolution in vivo optical imaging we have studied, at single-cell resolution, the spatio-temporal response of BMDCs in normal brain following CR. We demonstrate that BMDCs are recruited specifically to the site of CR, in a radiation dose and temporal-spatial manner. We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia. Most notably we provide evidence that more than 50% of the microglia in the irradiated region of the brain are not resident microglia but recruited from the bone marrow following CR. These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR. Identifying the critical steps involved in the sustained recruitment and differentiation of BMDCs into microglia at the site of CR can provide new insights into the mechanisms of injury following CR offering potential therapeutic strategies to counteract the long-term adverse effects of CR.

pone-0038366-g002: Bone Marrow Derived Cell Recruitment is Radiation Dose-Dependent.(A,B) Increasing levels of BMDC recruitment can be observed with increasing radiation doses on 2 photon laser microscopy in-vivo imaging 7 days post radiation (Green: BMDC, blue: CD31-APC), 10× magnification. (A) Single dose increase from 2Gy to 15Gy, (B) fractionated doses increase from 3*2Gy to 3*5Gy, all demonstrate an increase in BMDC recruitment. (C) Bar graph representing quantification of the extent of BMDCs recruited to site of cranial radiation 7 days post, demonstrating a statistically significant increase in BMDC recruitment between 2Gy to 6Gy and 2Gy to 15Gy, both p<0.0001***. Fractionated radiation demonstrates lower rate of BMDC recruitment when compared to its single fraction equivalent, perhaps explained by radiobiology principle of repair that occurs with fractionated radiation. The increase in BMDC recruitment is noted at all time points post radiation (1 hour, 1 day, 21 day).

Mentions:
Recruitment of BMDCs to the site of CR is radiation dose dependent. We delivered single doses of radiation, ranging from 2 to 15 Gy, plus two daily fractionated radiation regimens of 3×2Gy and 3×5Gy to the normal brain through the ICW. Using 2PLM imaging we demonstrated a visible increase in the extent of BMDC recruitment to the site of CR with radiation dose (Figure 2A,B), with the highest response seen at 15Gy at all time points post-RT. Quantifying the extent of BMDC recruitment demonstrates that with each higher radiation dose there is a statistically significant increase in BMDC recruitment (p<0.0001), with minimal BMDCs present on the contra-lateral left non-irradiated hemispheres (Figure 2C).

pone-0038366-g002: Bone Marrow Derived Cell Recruitment is Radiation Dose-Dependent.(A,B) Increasing levels of BMDC recruitment can be observed with increasing radiation doses on 2 photon laser microscopy in-vivo imaging 7 days post radiation (Green: BMDC, blue: CD31-APC), 10× magnification. (A) Single dose increase from 2Gy to 15Gy, (B) fractionated doses increase from 3*2Gy to 3*5Gy, all demonstrate an increase in BMDC recruitment. (C) Bar graph representing quantification of the extent of BMDCs recruited to site of cranial radiation 7 days post, demonstrating a statistically significant increase in BMDC recruitment between 2Gy to 6Gy and 2Gy to 15Gy, both p<0.0001***. Fractionated radiation demonstrates lower rate of BMDC recruitment when compared to its single fraction equivalent, perhaps explained by radiobiology principle of repair that occurs with fractionated radiation. The increase in BMDC recruitment is noted at all time points post radiation (1 hour, 1 day, 21 day).

Mentions:
Recruitment of BMDCs to the site of CR is radiation dose dependent. We delivered single doses of radiation, ranging from 2 to 15 Gy, plus two daily fractionated radiation regimens of 3×2Gy and 3×5Gy to the normal brain through the ICW. Using 2PLM imaging we demonstrated a visible increase in the extent of BMDC recruitment to the site of CR with radiation dose (Figure 2A,B), with the highest response seen at 15Gy at all time points post-RT. Quantifying the extent of BMDC recruitment demonstrates that with each higher radiation dose there is a statistically significant increase in BMDC recruitment (p<0.0001), with minimal BMDCs present on the contra-lateral left non-irradiated hemispheres (Figure 2C).

Bottom Line:
However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT.We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia.These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR.

ABSTRACTRadiation therapy (RT) is a widely accepted treatment strategy for many central nervous system (CNS) pathologies. However, despite recognized therapeutic success, significant negative consequences are associated with cranial irradiation (CR), which manifests months to years post-RT. The pathophysiology and molecular alterations that culminate in the long-term detrimental effects of CR are poorly understood, though it is thought that endothelial injury plays a pivotal role in triggering cranial injury. We therefore explored the contribution of bone marrow derived cells (BMDCs) in their capacity to repair and contribute to neo-vascularization following CR. Using high-resolution in vivo optical imaging we have studied, at single-cell resolution, the spatio-temporal response of BMDCs in normal brain following CR. We demonstrate that BMDCs are recruited specifically to the site of CR, in a radiation dose and temporal-spatial manner. We establish that BMDCs do not form endothelial cells but rather they differentiate predominantly into inflammatory cells and microglia. Most notably we provide evidence that more than 50% of the microglia in the irradiated region of the brain are not resident microglia but recruited from the bone marrow following CR. These results have invaluable therapeutic implications as BMDCs may be a primary therapeutic target to block acute and long-term inflammatory response following CR. Identifying the critical steps involved in the sustained recruitment and differentiation of BMDCs into microglia at the site of CR can provide new insights into the mechanisms of injury following CR offering potential therapeutic strategies to counteract the long-term adverse effects of CR.